Isolation and Culture of Human Liver Cells

This study was approved by the ethics committee of our institution. Liver tissue was obtained from a Ministry of Health-approved hospital tissue bank (Table 1). ADHLSCs were obtained after primary culture of the parenchymal fraction cells, as previously described [14 (link), 17 (link)]. The supernatant, containing the non-parenchymal cell fraction, was processed by Nycodenz gradient (Myegaard, Oslo, Norway) centrifugation to isolate HSCs [26 (link)]. Both cell types were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS; Life Technologies) and 1% penicillin/streptomycin (Life Technologies), at 37 °C in a humidified atmosphere containing 5% CO₂, as previously described [17 (link)]. When the cells reached 80% confluence, they were detached by treatment with 0.05% Trypsin-EDTA (Life Technologies). The viability of the recovered cells was evaluated using the trypan blue exclusion assay.Table 1

Characteristics of the four liver donors whose samples were used for the isolation of HSCs and ADHLSCs used in the current study

Donor number	Age	Gender	Cause of death	Blood group	Ischemia time
89	3 days	M	Respiratory	A+	4 h
93	2 years	F	Metabolic disease (liver transplanted)	O+	1 h 43 min
98	7 days	M	Cardio-respiratory arrest	O-	4 h 20 min
105	46 years	F	Trauma	B+	9 h 43 min